Force-triggered rapid microstructure growth on hydrogel surface for on-demand functions

Living organisms share the ability to grow various microstructures on their surface to achieve functions. Here we present a force stamp method to grow microstructures on the surface of hydrogels based on a force-triggered polymerisation mechanism of double-network hydrogels. This method allows fast spatial modulation of the morphology and chemistry of the hydrogel surface within seconds for on-demand functions. We demonstrate the oriented growth of cells and directional transportation of water droplets on the engineered hydrogel surfaces. This force-triggered method to chemically engineer the hydrogel surfaces provides a new tool in addition to the conventional methods using light or heat, and will promote the wide application of hydrogels in various fields.

While the applications are nice, I think the greatest impact of the paper lies in the core principle, which basically takes a perceived weakness of hydrogels (their mechanical fracture) and tames it to make it useful. I think this concept and its extension will be inspiring to materials scientists, polymer chemists, biomedical engineers, and mechanical engineers who might think of coupling instabilities to lithography to create other patterns. The writing was quite easy for me to follow and seems suitable to Nat. Commun. Finally, the videos are quite compelling (direct links from the online MS would be a nice touch).
I endorse publication following one scientific adjustment and a few edits for clarity: Science --the authors make a point of emphasizing the reduction in "second network outer layer" for successful surface patterning. but the process zones used to initiate response are often hundreds of microns (per the authors) and the surface layer only 1-10 microns thick. does the use of a hydrophobic mold and reduced surface layer really matter? I assume so, and that the authors have evidence of at least some isolated issues faced when using the conventional preparation methods. It would be useful to share an example or examples of cases where the surface layer has an actual impact, or to state explicitly that it is only a hypothesis that the surface layer would have a detectable impact on the technique.
Grammatical corrections --Page 3. "Furthermore, the top-most surface of a conventional DN hydrogel is covered by a layer of the soft second network with 1−10 μm-thick. " should be "Furthermore, the top-most surface of a conventional DN hydrogel is covered by a layer of the soft second network [that is approximately] 1−10 μm-thick." Page 4. "The internal fracture of the brittle network is proportional to the macroscopic deformation and spatially controllable and the concentration of mechanoradicals..." could be "The internal fracture of the brittle network is [both] proportional to the macroscopic deformation and spatially [controllable. The] concentration of mechanoradicals..." "and of its second network is polyacrylamide (PAAm)." should be "and [whose] second network is polyacrylamide (PAAm)." "...covered by a nonionic second network layer of several micrometers thick..." should be "...covered by a nonionic second network layer [that is] several micrometers thick..." Page 6. "by covalent bonds rupture of the first network" could be "by the rupture of covalent bonds in the first network" "the mechanoradicals concentration " should be "the [mechanoradical] concentration" Reviewer #2: Remarks to the Author: In this paper, Nakajima and Gong et al demonstrated the force-induced microstructure growth on the surface of double-network hydrogels. They successfully realized the homogeneous bond breaking on the surface of double-network hydrogels to afford mechano-radicals that can polymerize free monomers in the gels. The spatially controllable structural change can bring drastic changes in surface chemistry and topology for functional change. Actually, oriented cell growth and directional water transportation were successfully demonstrated. I think the present results will contribute to the progress of mechanochemistry, polymer chemistry, surface chemistry, and materials science. The importance of this paper is high enough to warrant publication in Nature Communications, but the revision of the following minor points is required before publication. 1) Using a hydrophobic mold has created a double network structure on the top-most surface layer. When a hydrophilic mold is used, does the polymerization also occur in gels? Several micrometers are covered by a second network layer, whereas the indentation scale is 1000 micrometers. In that case, do the surface properties remain the same?
2) Why is the polymerization rate of NIPAM and NaAMPS significantly different in Supplementary Figure S6? Are the stability of the growing radicals or the diffusion rate of the monomers affected?
3. Since the NIPAM showing LCST is polymerized in Figures 2, 3, and S20, the temperature should be shown. Figure 3c, only NIPAM is non-ionic. Even if the degree of polymerization is the same, the degree of swelling may be lower. 5. Line 258: "bulk DN hydrogel is composed of thermal and pH-insensitive polymer," Does protonation of NaAMPS units affect the properties of DN gels? 6. PNIPAM should be able to detach cells depending on temperature, but does this system detach?

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Response to Reviewers
Response to Reviewer #1 I very much enjoyed this paper, which builds off a landmark paper by many of these authors (ref. 19). The use of mechanical force to activate chemical bond formation (polymerization) is quite interesting and is nicely distinct from other forms of lithography, many of which are not suitable to hydrogel patterning. The result has reasonable generality, as demonstrated by the authors here. The level of control and resolution is sufficient to drive some applications, in particular the cell patterning and water transport examples provided.
While the applications are nice, I think the greatest impact of the paper lies in the core principle, which basically takes a perceived weakness of hydrogels (their mechanical fracture) and tames it to make it useful. I think this concept and its extension will be inspiring to materials scientists, polymer chemists, biomedical engineers, and mechanical engineers who might think of coupling instabilities to lithography to create other patterns. The writing was quite easy for me to follow and seems suitable to Nat.
Commun. Finally, the videos are quite compelling (direct links from the online MS would be a nice touch).
Answer: Dear reviewer, we sincerely thank you for your time and effort in reviewing our paper. We have revised the manuscript after carefully considering your comments and suggestions. All modifications are shown in red in the revised manuscript.
I endorse publication following one scientific adjustment and a few edits for clarity: 1. Science --the authors make a point of emphasizing the reduction in "second network outer layer" for successful surface patterning. but the process zones used to initiate response are often hundreds of microns (per the authors) and the surface layer only 1-2 matter? I assume so, and that the authors have evidence of at least some isolated issues faced when using the conventional preparation methods. It would be useful to share an example or examples of cases where the surface layer has an actual impact, or to state explicitly that it is only a hypothesis that the surface layer would have a detectable impact on the technique.
Answer: Thanks for your precious comments. Yes, we should make this important point clearer. As you commented, since our micro indentation causes internal fracture deeper than the surface layer, radical polymerization occurs in the damaged region in response to mechanical indentation regardless of the presence or absence of a surface layer.
However, in the presence of a surface layer, no mechanoradical polymerization occurs on the soft surface layer. Therefore, to have the chemical modification of the hydrogel surface, it is required to use DN hydrogels without the soft surface layer.
To justify this argument, we added a result for a DN hydrogel with a soft surface layer.
We performed the indentation of the DN hydrogel immersed in NIPAm solution. We "and of its second network is polyacrylamide (PAAm)." should be "and [whose] second network is polyacrylamide (PAAm)." "...covered by a nonionic second network layer of several micrometers thick..." should be "...covered by a nonionic second network layer [that is] several micrometers thick..." Page 6. "by covalent bonds rupture of the first network" could be "by the rupture of covalent bonds in the first network" "the mechanoradicals concentration " should be "the [mechanoradical] concentration" Answer: Thanks for your helpful advice. We corrected the grammatical errors in page 3, 4, 6 in accordance with your suggestions.

Response to Reviewer #2
In this paper, Nakajima and Gong et al demonstrated the force-induced microstructure growth on the surface of double-network hydrogels. They successfully realized the homogeneous bond breaking on the surface of double-network hydrogels to afford mechano-radicals that can polymerize free monomers in the gels. The spatially controllable structural change can bring drastic changes in surface chemistry and topology for functional change. Actually, oriented cell growth and directional water transportation were successfully demonstrated. I think the present results will contribute to the progress of mechanochemistry, polymer chemistry, surface chemistry, and materials science. The importance of this paper is high enough to warrant publication in Nature Communications, but the revision of the following minor points is required before publication.
Answer: Dear reviewer, we sincerely thank you for your time and effort in reviewing 4 our paper. We have revised the manuscript after carefully considering your comments and suggestions. All modifications are shown in red in the revised manuscript.
1. Using a hydrophobic mold has created a double network structure on the top-most surface layer. When a hydrophilic mold is used, does the polymerization also occur in gels? Several micrometers are covered by a second network layer, whereas the indentation scale is 1000 micrometers. In that case, do the surface properties remain the same?
Answer: Thanks for your precious comments. Yes, we should make this important point clearer. As you commented, since our micro indentation causes internal fracture deeper than the surface layer, radical polymerization occurs in the damaged region in response to mechanical indentation regardless of the presence or absence of a surface layer.
However, in the presence of a surface layer, no mechanoradical polymerization occurs on the soft surface layer. Therefore, to have the chemical modification of the hydrogel surface, it is required to use DN hydrogels without the soft surface layer.
To justify this argument, we added a result for a DN hydrogel with a soft surface layer.
We performed the indentation of the DN hydrogel immersed in NIPAm solution. We confirmed the absence of surface chemical modification but the presence of polymerization in the region beneath the surface layer. We added this result in Supplementary Figures 15 and 16, and more explanation in the revised manuscript (page 8, lines 224-233).

Why is the polymerization rate of NIPAM and NaAMPS significantly different in
Supplementary Figure S6? Are the stability of the growing radicals or the diffusion rate of the monomers affected?
Answer: Thanks for your comments. The difference in polymerization rate is an interesting phenomenon (shown in Supplementary Figure 6). The polymerization rate of monomers in DN hydrogels can be affected by various factors, such as the diffusion 5 rate of monomers and growing chains, radical stability, the interaction between the monomers/growing chains with the hydrogel networks, etc. Currently, we do not have enough experimental evidence to make clear conclusions. We would like to further investigate how the polymerization rate is related to these factors in future work.
3. Since the NIPAM showing LCST is polymerized in Figures 2, 3, and S20, the temperature should be shown.
Answer: Thanks for your comments. We have added the temperature information in We agree that NaAMPS units can be partly protonated in acidic solution. To verify whether the volume and properties of DN hydrogels can be affected by the protonation, we immersed the DN hydrogel into acid and alkaline solutions ( Figures R1, R2). The results reveal that pH has no effect on swelling ratio of the DN hydrogel, suggesting effect of protonation of NaAMPS units on the properties of DN hydrogels is negligible.
It has been known that swelling ratio of DN hydrogels is dominated by the dense nonionic PAAm network owing to its high osmotic pressure, not by the sparse PNaAMPS network (Nakajima, T. et al. Soft Matter, 16, 5487-5496 (2020)). Even though NaAMPS units are possibly protonated at low pH, such structure change on the PNaAMPS network does not affect the swelling and related properties of DN hydrogels because of the presence of the dense PAAm network.
The sentence pointed out by the reviewer has been changed as: "Since the bulk DN hydrogel is insensitive to heat or pH change, the induced surface microstructures could be controlled by these stimuli without altering bulk properties."  6. PNIPAM should be able to detach cells depending on temperature, but does this system detach?
Answer: Yes. We have preliminary found that the C2C12 cells attach on the PNIPAMpatterned DN hydrogel at 37 °C ( Figure R3 a) and automatically detach at 5 °C ( Figure   R3 b). Since the scope of this paper is developing a novel hydrogel surface engineering method, we will report this temperature effect in a separate paper. Figure R3. Thermoresponsive adhesion and detachment of C2C12 cells on the PNIPAM patterned hydrogels. a. At 37 o C, most cells significantly adhered to the PNIPAM pattern. b. At 5 o C, the cell clusters detached from the PNIPAM pattern automatically. Cells were incubated at 5 °C for 5 hours prior to the microscopic observation. The dotted circles mark the detached cell clusters, the red arrows show the possible detachment paths.